Introduction

The protection of neurons from damage and death is an important challenge in the development of treatment of brain ischemia and neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Cortical neurodegeneration in Alzheimer’s disease and dopaminergic neurodegeneration in Parkinson’s disease may be attributed, at least in part, to neurotoxicity induced by glutamate (Hynd et al. 2004; Martinez et al. 1993). Glutamate acts as a neurotoxic substance when it is present in excess. Glutamate is postulated to play a crucial role in the neurodegeneration observed in hypoxic–ischemic brain injury in addition to its role in neurotransmission (Meldrum and Garthwaite 1990). The mechanism of glutamate neurotoxicity has been extensively studied, and it has been widely accepted that glutamate induces neuronal death via stimulation of N-methyl-D-aspartate (NMDA) receptor through which Ca2+ enters the cell and activates Ca2+-dependent nitric oxide synthase, resulting in excessive nitric oxide formation, production of radicals, mitochondrial dysfunction, and cell death (Dawson et al. 1991; Tamura et al. 1992).

The actions of glutamate as an excitatory neurotransmitter are regulated by many other endogenous substances, such as inhibitory neurotransmitters and neuromodulators. Suppression of the control on glutamate-induced excitation causes severe dysfunction of central nervous system (CNS) activity. Thus, excitatory actions of glutamate and other excitatory amino acids should be tonically regulated by other substances to maintain normal neuronal activities in CNS. Meanwhile, the cytotoxic effects of excitatory amino acids, including glutamate, have a crucial role in the pathogenesis in the neurodegeneration. Moreover, most CNS neurons express glutamate receptor subtypes including NMDA receptors. This suggests that neurons in CNS are exposed to both excitatory and cytotoxic effects of glutamate. Then, it is hypothesized that neurotoxic action of glutamate is also regulated by other endogenous substances in patho/physiological conditions (Akaike 2006; Kume et al. 2002). In other words, certain substances including neurotransmitters may possess neuroprotective action against glutamate cytotoxicity to promote neuronal survival in CNS.

Alzheimer’s disease is a progressive neurodegenerative disorder characterized by the depletion of high affinity nicotinic acetylcholine receptors (nAChR) and marked loss of cholinergic neurons (Collerton 1986; Whitehouse et al. 1982). Donepezil, galantamine, and tacrine are acetylcholinesterase (AChE) inhibitors that have been developed for treatment of Alzheimer’s disease under a presumption that increasing cholinergic neurotransmission through inhibition of AChE may enhance cognitive function. Although the role of glutamate neurotoxicity in Alzheimer’s disease is not yet clear, it has been reported that amyloid β protein (Aβ) enhances vulnerability of neurons to glutamate neurotoxicity, indicating that glutamate may play important roles in Aβ-induced cytotoxicity in the cerebral cortex (Kihara et al. 1997). Furthermore, many previous studies have demonstrated that persistent stimulation of certain subtypes of nicotinic acetylcholine receptors (nAChRs), such as α4 and α7, protect against neurotoxicity induced by glutamate and Aβ (Akaike et al. 1994; Kaneko et al. 1997; Kihara et al. 1997; Rosa et al. 2006). Persistent stimulation of nAChRs is also known to up-regulate the expression level and some functions of nAChRs, and this effect is often associated with desensitization. However, the results of these analyses are not yet conclusive, likely due to multiple desensitized states, differences in kinetics, or ligand-specific differences, and the functional state of up-regulated nAChRs in vivo is still controversial (Takada-Takatori et al. 2009). This review aim to introduce the recent advances in the studies on mechanisms of neuroprotection and nAChR up-regulation induced by nicotine and AChE inhibitors.

Nicotine-Induced Neuroprotection via α4 and α7 nAChRs

On exposure to the agonists, nAChR exerts an active, open state and elicits rapid depolarization. The progressive decline of the agonist-evoked current reveals the closure of the channel, and nAChR exerts desensitized, non-functional states. Moreover, it has been widely recognized that nAChRs mediate the long-term modification of cell functions via specific signaling systems as well as the short-term excitation of the cells (Dajas-Bailador and Wonnacott 2004). Neuronal nAChRs are also recognized to facilitate cell survival upon neurotoxic insults. Persistent stimulation of nAChR protects CNS neurons against cytotoxicity induced by glutamate and Aβ (Kihara et al. 1997; Shimohama et al. 1996). The studies by Akaike et al. (1994) and Kaneko et al. (1997) clearly demonstrated the protective effect of nicotine against glutamate neurotoxicity via nAChR subtypes expressed in the CNS. A characteristic feature of neuroprotective action of nicotine was that a relatively long exposure (8–24 h) was necessary to ameliorate glutamate neurotoxicity. It is possible that prolonged nicotine treatment causes desensitization of nAChRs to cause receptor dysfunction, which may be a dominant mechanism of nicotine-induced neuroprotection. However, this possibility is unlikely since the protective action of nicotine is clearly antagonized by selective antagonists of nAChRs, mecamylamine, methyllicaconitine (selective for α7 nAChR), and dihydro-β-erythroidine (selective for α4 nAChR). Those findings indicate that nicotine protects CNS neurons against glutamate neurotoxicity by facilitating functions of α4 and α7 nAChRs.

In CNS, nAChRs include several subtypes with different properties and functions. The abundant presence of α7 nAChRs in the hippocampus and neocortex, in conjunction with the memory-enhancing activity of selective α7 nAChR agonists, suggests a significant role of α7 nAChRs in learning and memory (Levin et al. 2006). In addition, the protective action of nicotine is mediated, at least partially, through α7 nAChRs. A study by Kihara et al. (2001) has shown that nAChR stimulation, especially α7 receptor stimulation, inhibits glutamate neurotoxicity and that phosphatidylinositol 3-kinase (PI3K) signal transduction contributes the neuroprotective effect (Fig. 1). In this study, neuroprotective effect of nicotine was reduced by PI3K inhibitors, LY294002 and wortmannin, and a non-receptor tyrosine kinase inhibitor, PP2. Nicotine increased the level of phosphorylated Akt, an effector of PI3K, and Bcl-2 detected by an antibody specific for phospho-Akt using Western blotting, and its effect was blocked by a PI3K inhibitor. Physical association of a7 nAChR, PI3K, and Fyn, a non-receptor tyrosine kinase, was confirmed by immunoprecipitation study. Bcl-2 proteins are anti-apoptotic proteins, which protect cell death induced by toxic insults. It has been reported that Akt activation leads to the overexpression of Bcl-2 (Matsuzaki et al. 1999). Thus, we examined the protein level of Bcl-2 and demonstrated that nicotine treatment for 24 h induced the augmented level of Bcl-2 and that the nicotine-induced up-regulation of Bcl-2 was reduced by a PI3K inhibitor. These observations led us to assume that nAChR stimulation facilitates phosphorylation of Akt by signal transduction through Fyn and PI3K. Ca2+ influx through nAChRs might contribute to this process.

Figure 1
figure 1

A scheme showing the mechanism of neuroprotective effect of nicotine via α7 nAChR in cultured cortical neurons

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Neuroprotective Effects of AChE Inhibitors

Direct evidence of neuroprotection by AChE inhibitors came from studies on the effects of donepezil in oxygen–glucose deprivation model of ischemia using rat PC12 cells and rat primary neuronal culture of the cerebral cortex (Zhou et al. 2001; Akasofu et al. 2003). Around the same period, neuroprotective effects of AChE inhibitors against glutamate neurotoxicity were discovered by our group in a rat primary culture of cerebral neurons (Takada-Takatori et al. 2003). In this study, pretreatment with donepezil for 24 h significantly inhibited glutamate-induced loss of viability. On the other hand, simultaneous treatment with donepezil and glutamate did not protect neurons from neurotoxicity. Donepezil, galantamine, and tacrine also protected against neuronal death induced by moderate glutamate treatment-induced neuronal death that is associated with apoptosis (Takada-Takatori et al. 2006a, b). Protective effects of pretreatment with six other AChE inhibitors were tested, and not only donepezil but also galantamine and tacrine as well as neostigmine and pyridostigmine also protected against glutamate neurotoxicity. However, physostigmine, albeit the strongest potency among the AChE inhibitors tested, did not protect against glutamate. Donepezil, galantamine, and tacrine significantly protected against glutamate neurotoxicity in a concentration-dependent manner from 0.1 to 10 μM, a clinically relevant physiologic concentration. These concentrations were higher than their half-maximal inhibitory concentration, suggesting that the neuroprotective effects of AChE inhibitors were not due the AChE inhibition.

Involvement of nAChR in nicotine-induced neuroprotection against glutamate neurotoxicity had been indicated for some time prior to our study on the mechanism of AChE inhibitor-induced neuroprotection (Akaike et al. 1994; Kaneko et al. 1997). Furthermore, preclinical electrophysiological experiments proposed that galantamine potentiates ligand action on nAChR possibly through allosteric modulation of nAChR (Maelicke et al. 2001). Several studies indicate that donepezil may also interact and regulate nAChR function, although the results were not conclusive (Nordberg 2006). Thus, we examined the effects of nAChR antagonists on AChE inhibitor-induced neuroprotection and observed that nAChR antagonists but not muscarinic receptor antagonist inhibits neuroprotection against glutamate neurotoxicity (Takada-Takatori et al. 2003). The antagonistic effects of dihydro-β-erythroidine and methyllycaconitine, inhibitors of α4 and α7 nAChRs, respectively, suggest participation of these receptor subtypes in donepezil- and galantamine-induced neuroprotection. Neuroprotection by tacrine was inhibited by mecamylamine but not subtype selective antagonists, suggesting the involvement of other nAChR subtypes in the neuroprotection.

The observations indicating the involvement of nAChRs in AChE-inhibitor-induced neuroprotection led us to examine the involvement of PI3K-Akt-Bcl-2 pathway in AChE inhibitor-induced neuroprotection. Upon stimulation by nicotine, α7 nAChR activates PI3K and promotes survival of neuronal cells via activation of Akt-Bcl-2 pathway (Kihara et al. 2001). For AChE inhibitors, treatment with PP2, AG490, LY294002, and wortmannin, inhibitors of Fyn, janus-activated kinase 2 (JAK2), and PI3K, respectively, significantly inhibited neuroprotection by donepezil and galantamine but not tacrine, which is in good accordance with the result of treatment with subtype selective antagonists described above. The level of phosphorylated Akt increases upon nicotine treatment and is suppressed with PI3K inhibitor or JAK2 inhibitor treatment. Activation of Akt in turn increases the expression level of Bcl-2 transcript. We observed that treatment with donepezil and galantamine but not tacrine also increases the phosphorylation level of Akt and the expression level of Bcl-2 transcript (Takada-Takatori et al. 2006a,b). Thus, the mechanism of AChE-inhibitor-induced neuroprotection inferred from our results is as follows: donepezil and galantamine facilitate functions of nAChRs, especially α7 nAChR, either directly or indirectly and trigger activation of PI3K-Akt-Bcl-2 pathway to promote neuronal survival (Fig. 2). Regarding tacrine, nAChR subtypes involved in tacrine-induced neuroprotection and the intracellular mechanisms involved are not clear.

Figure 2
figure 2

A scheme showing the mechanisms of neuroprotective effects of AChE inhibitors, donepezil, galantamine, and tacrine, in cultured cortical neurons

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Up-regulation of nAChRs by nicotine and AChE inhibitors

It is well known that chronic nicotine treatment up-regulates nAChRs. In case of AChE inhibitors, several clinical and preclinical studies demonstrated that administration of donepezil or galantamine increased the level of nAChR in rat and human brains (Barnes et al. 2000; Reid and Sabbagh 2003; Nordberg et al. 1998). Thus, we examined the possibility that AChE inhibitors may induce up-regulation of nAChRs like nAChR agonists and observed significant up-regulation of α4 and α7 nAChR protein level with longer treatment, after 4 days chronic treatment with donepezil (Takada-Takatori et al. 2008a,b). We also observed significant increase in the proportion of neurons expressing α4 and α7 nAChR after chronic treatment with donepezil and galantamine. Prolonged treatment of cortical cultures with donepezil, galantamine, or tacrine potentiated the nicotine-induced increase in intracellular calcium concentration ([Ca2+]i), and no signs indicating desensitization of nAChRs were detected upon AChE treatment. Similarly, a 4-day treatment of cortical cultures with nicotine itself potentiated nicotine-induced increase in [Ca2+]i.

The mechanism of nAChR up-regulation by nicotine has been extensively studied using cell lines stably expressing α4 or α7 nAChRs. These studies suggested that nAChRs are up-regulated by post-translational mechanisms involving receptor stabilization, changes in endocytic trafficking and receptor turn-over, exocytic trafficking of nAChRs from the endoplasmic reticulum to the cell surface, or enhanced intracellular maturation through chaperone function of nicotine (Darsow et al. 2005; Sallette et al. 2005; Kuryatov et al. 2005; Marks et al. 1992). We also examined the mechanism of donepezil-induced nAChR up-regulation using primary culture of rat cortical neurons (Takada-Takatori et al. 2008a,b). Chronic treatment with donepezil did not alter the level of mRNAs of α4 and α7 subunits examined by reverse transcription polymerase chain reaction, demonstrating that donepezil up-regulates nAChR by post-translational mechanisms. Then, using specific inhibitors, we investigated the involvement of signal transduction pathway linked with PI3K in the up-regulation of nAChR. Treatment with PP2, AG490, and LY294002 inhibited α7 nAChR up-regulation upon chronic donepezil treatment, suggesting the involvement of α7 nAChR-PI3K pathway. Furthermore, donepezil-induced up-regulation was significantly inhibited by treatment with PD98059, an inhibitor of MAPK. Donepezil-induced functional up-regulation of nAChR, as shown by the enhancement of nicotine-induced increase of [Ca2+]i also significantly inhibited by PP2, AG490, LY294002, and PD98059. These results indicate that functional up-regulation of nAChR may be due to the increase in nAChRs at the cell surface. Another point of interest is how galantamine and tacrine functionally up-regulates nAChR without apparent increase in nAChR density. Up-regulation by galantamine likely involves allosteric modulation of nAChR function. On the other hand, mechanism of tacrine-induced up-regulation is not clear, and it is not known whether tacrine physically interacts with nAChR and modulate its functions.

Conclusion

Many studies have established the protective effect of nicotine against neuronal death induced by glutamate, Aβ, and other toxic insults. One of the first studies to clearly demonstrate that persistent stimulation of nAChRs promotes survival of neurons under stress by exogenous glutamate was Akaike et al. in 1994. Thereafter, nAChR have attracted attentions of many scientists because of their neuroprotective properties and their involvement in pathologies of Alzheimer’s disease and many other neurodegenerative diseases. We and several others have demonstrated the involvement of α4 and α7 nAChRs in neuroprotection induced by nicotine and centrally acting AChE inhibitors, donepezil and galantamine. AChE inhibitors also induced up-regulation of α4 and α7 nAChRs, and it has been shown that nAChRs and intracellular signaling pathway involving PI3K and MAPK play important roles in both neuroprotection and up-regulation. A long-lasting belief for AChE inhibitors used for the treatment of Alzheimer’s disease is that those agents are purely symptomatic and work by slowing the hydrolysis of ACh at CNS terminals. However, neuroprotective effects of AChE inhibitors, donepezil and galantamine, seem to be independent of their ability on hydrolysis of ACh. The neuroprotective effects of clinically relevant concentrations of AChE inhibitors may be due to a unique aspect of their pharmacology. Further studies elucidating the precise mechanisms of neuroprotective effects via nAChRs would lead to novel strategies in the prevention and treatment of neurodegenerative diseases.